Matrix-assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry For Resistant Bacterial Strain Detection And Typing Of Vibrio Cholerae

Objective:To explore the differences between non-resistant strains and resistant strains in their protein fingerprint spectra by matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF MS)technology and find out the impact factors of protein profiles, which lays a foundation for establishing drug resistance bacteria protein fingerprint database in the future. Moreover,192strains of Vibrio choleraes were collected from our representative cities and counties in Zhejiang province.All strains of Vibrio choleraes were characterized by routine serological typing,16S rRNA sequence analysis and MALDI-TOF MS analysis. The aim is to reveal the changes of serum type of Vibrio cholerae isolates in Zhejiang Province from1962to2009and to compare, at a methodological, the discriminatory powers of16S rRNA and MALDI-TOF MS for Vibrio cholerae, and to provide effective tools for further study on the evolution rule of Vibrio cholerae strains and such data are critical for understanding why and how pandemic-causing Vibrio, cholerae isolates.Methods (1) The single drug-resistant strains and multi drug resistant bacteria of the same strain was performed by MALDI-TOF MS technology.(2) The logarithmic growth phase of single colony of resistance bacterial was choosed and treated by ethanol/formic acid and sensitive strains was used as negative control, Their protein fingerprint profiles were detected by MALDI-TOF MS.(3)192strains of Vibrio cholera from Zhejiang province were processed by routine serological typing,16S rRNA sequence analysis, and MALDI-TOF MS analysis.Results:(1) The bacterial protein has a good repeatability between the repeats. Similar protein fingerprint were achieved from the same bacteria strains, indicating that the protein expression was constant between the same resistant bacteria strains, Incubation time has slight influence on bacterial protein fingerprint, but the best time for sampling was logarithmic growth phase of bacteria. A simulation experiment was carried out with88strains of multi drug resistant strains, the results showed MALDI-TOF MS cannot effectively distinguish the resistance strains and sensitive strains. But some factors that affect the drug-resistant bacteria protein peaks were found, including growth phase of bacteria and sample pretreatment method. There was a slight influence to fingerprint acquisition with direct smear approach target without pretreatment for gram negative. However, the influence is great to Gram positive bacteria.In addition, Although it was not divided effectively between drug-resistant bacterial and non-resistant strains by MALDI-TOF MS, the study had established the protein fingerprint database of these strains and expanded the MALDI-TOF MS database, which laid the foundation for the identification of bacteria.(2) The dendrogram of MALDI-TOF MS, the protein fingerprint of Vibrio cholerae samples were divided into two categories, the cluster was very obvious, the red part of the dendrogram above was also divided into two parts. In the16S rRNA clustering analysis, samples were divided into two categories, one was divided into a and b.the other is c branch11In the MALDI-TOF MS clustering analysis, the c class was divided into V009(1965/Inaba type), V164(1995/Ogawa), V045(2000/Inaba) in the branch and8Vibrio cholerae samples were classified as in the other branch, Results of two methods were basically the same, However, MALDI-TOF MS with higher resolution and the degree of differentiation, but the two methods were not exactly consistent in the serum level. From the classification results, The serotype was related to years. There were35strains of Ogawa among a collection of38strains that accounted for96.5%from1995to1999.Inaba serotype can been seen in the late1970s and from2000to2005. Ogawa serotype aslo been found, but it was not the major epidemic strains. Ogawa serotype was major strains in2008or2009. There was a correlation between the serum type strains and places of isolation, However, it was not necessarily associated with places of isolation. The cholera outbreaks usually occurred in the coast of Zhejiang. Beside, seafood was more convenient for transportation than before. If a place happened epidemic cholera, it would cause another inland became popular in the short time because of convenient transportation.Conclusion: The matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) cannot distinguish remarkably difference of protein fingerprint profiles of drug-resistant bacteria and these of non drug-resistant bacteria. However, the protein fingerprints of these resistant strains have added the bacteria identification database,which laid the foundation for the identification of bacteria.192strains of Vibrio cholerae from Zhejiang province were analysised by16S rRNA sequence analysis, and MALDI-TOF MS analysis, the results revealed the changes of serum type from1962to2009in Zhejiang Province, provideing some useful data and information for further research on the evolution rule of Vibrio cholera isolates in Zhejiang province.